Conserved Role of the Linker α-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB

Hiram Gilbert,George Georgiou,Laura Segatori,Hiroshi Kadokura,Jon Beckwith,Silvia Arredondo,Lori Murphy

doi:10.1074/jbc.m505453200

Abstract

In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the alpha-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying alpha-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved.

Highlights

Disulfide bonds formation is a critical step in protein folding
Because 3.6-amino acidic residues are contained in a ␣-helical turn, the deletion of each amino acid in the DsbC linker would be expected to cause the catalytic domain to be rotated by 100° with respect to the dimerization domain
Under the conditions used in these experiments, the wildtype E. coli DsbC and the ␣-helix deletion mutants all accumulated to nearly identical levels, as determined by Western blotting with a polyclonal antibody specific for the C-terminal His tag (Fig. 2A)

Summary

Introduction

Disulfide bonds formation is a critical step in protein folding. By covalently cross-linking amino acids far apart in the protein primary structure, the formation of native disulfides is associated with the increased stability and structural complexity typical of many secreted proteins. In DsbG as well as in the DsbCs from both E. coli and Haemophilus influenza [9], the ␣-helical linker serves to place the CXXC active sites within the two thioredoxin domains directly facing each other, indicating that this is a conserved feature of bacterial disulfide isomerase enzymes. These observations, together with our data using DsbC chimeras [7], suggested that the ␣-helical linker may play a pivotal role in the function of DsbC enzymes. The DsbC mutant enzymes retained the ability to catalyze the rearrangement of disulfide bonds in substrate proteins expressed in the E. coli periplasm, indicating that the reduction of the enzyme by DsbD is not compromised by the deletions

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Results

Conclusion